Adrenomedullin (AM) is a recently identified hypotensive peptide initially isolated from human pheochromocytoma (K. Kitamura, et al., Biochem. Biophys. Res. Commun. 192, 553 (1993)). AM is generated from a 185 amino acid preprohormone through consecutive enzymatic cleavage and amidation. This process culminates in the liberation of a 52 amino acid bioactive peptide (T. Ishimitsu et al., Biochem. Biophys. Res. Commun. 203, 631 (1994)). AM and its gene-related peptide, PAMP, are the two known bioactive products generated from the post—translational enzymatic processing of the 185 amino acid-preproAM molecule (K. Kitamura, et al., Biochem. Biophys. Res. Commun. 192, 553 (1993); K. Kitamura, et al., Biochem. Biophys. Res. Commun. 194, 720 (1993); Kitamura, et al. FEBS Lett. 351, 35-37 (1994)).
The complete genomic infrastructure for human AM has recently been reported (Ishimitsu, et al., Biochem Biophys Res Commun 203:631-639 (1994)). The porcine (Kitamura, et al., FEBS Lett 338:306-310 (1994)) and rat (Sakata, et al., Biochem Biophys Res Commun 195:921-927 (1993)) AM complementary DNAs have also been cloned/sequenced and demonstrate high homology to the human counterpart. Human cDNA of AM has been cloned and mRNA expression identified in the adrenal glands, lung, kidney, and heart (K. Kitamura, et al., Biochem. Biophys. Res. Commun. 194, 720 (1993)). A high degree of base sequence homology has been found between AM mRNAs isolated from other mammalian species, including rat and porcine (J. Sakata, et al., Biochem. Biophys. Res. Commun. 195, 921 (1993); and K. Kitamura, et al., FEBS Lett. 338, 306 (1994)).
Data from several publications have demonstrated a wide range of tissues that express AM. Using RIA and Northern blot techniques, high levels of AM have been found in human plasma, adrenal medulla, heart atrium, lung, and kidney (Kitamura, et al., Biochem Biophys Res Commun 194:720-725 (1993); Kitamura, et al., FEBS Lett 341:288-290 (1994)), but, to date, the cell source of AM in these organs has not been identified.
Although both AM and PAMP are amidated peptides, they have been shown to mediate their vasodilatory effects through distinctly different receptor systems (T. Shimosawa, et al., J. Clin. Invest. 96, 1672 (1995)). AM stimulates adenyl cyclase activity, which elevates cAMP levels in smooth muscle cells. AM is structurally related to calcitonin gene-related peptide (CGRP), and its vasodilatory effect is inhibited by the CGRP antagonist, CGRP8-37 (Y. Ishiyama, et al., Eur. J. Pharmacol. 241, 271 (1993); Ishizaka, et al., Biochem. Biophys. Res. Commun. 200, 642 (1994); J. A. Santago, et al., Life Sci. 55, 85 (1994); D. Y. Cheng, et al., Life Sci. 55, 251 (1994); H. Lippton, et al., J. Appl. Physiol. 76, 2154 (1994); Y. Shimekake, et al., J. Biol. Chem. 270, 4412 (1995)). Conversely, the fact that PAMP has no amino acid sequence homology to CGRP and its biological effects are not blocked by CGRP8-37 suggests the involvement of a separate receptor system (T. Shimosawa, et al., J. Clin. Invest. 96, 1672 (1995)). AM has also been implicated as an important regulator of renal function having natriuretic and diuretic action (T. Ebara, et al., Eur. J. Pharmacol. 263, 69 (1994); M. Jougasaki, et al., Amer. J. Physiol. 37, F657 (1995)). AM is also reported to be a potent bronchodilator, a regulator of certain central brain actions (vasopressor and antidipsogenic peptide), and a suppressor of aldosterone and adrenocorticotropin release (H. Kanazawa, et al., Biochem. Biophys. Res. Commun. 205, 251 (1994); H. Takahashi, et al., Am. J. Hypertens. 7, 478 (1994); T. A. Murphy and W. K. Samson, Endocrinology 136, 2459 (1995); T. Yamaguchi, K. Baba, Y. Doi, K. Yano, Life Sci. 56, 379 (1995); W. K. Samson, T. Murphy, D. A. Schell, Endocrinology 136, 2349 (1995)). Finally, AM has been shown to be expressed in a variety of human tumors of both neural and pulmonary lineage including ganglioblastoma/neuroblastoma (F. Satoh, et al., J. Clin. Endocrinol. Metabol. 80, 1750 (1995)), small cell lung cancer, adenocarcinoma, bronchoalveolar carcinoma, squamous cell carcinoma, and lung carcinoids (Martínez, et al., Endocrinology 136, 4099 (1995)). In an attempt to further study the distribution of AM in human tumors and determine its role in these malignant disorders, we used molecular, biochemical and in vitro techniques to analyze 59 human cancer cell lines from solid tumors and hemopoietic lineage.
AM's role as a vasodilatory agent has been extensively studied (C. Nuki et al., Biochem. Biophys. Res. Commun. 196, 245 (1993); Q. Hao et al., Life Sci. 54, 265 (1994); D. Y. Cheng et al., Life Sci., 55, 251 (1994); C. J. Feng, B. Kang, A. D. Kaye, P. J. Kadowitz, B. D. Nossaman, Life Sci., 433 (1994)). It acts through specific receptors in the plasma membrane to activate adenylate cyclase activity and modulate Ca2+ flux in the target cells (S. Eguchi et al., Endocrinology 135, 2454 (1994); Y. Shimekake et al., J. Biol. Chem. 270, 4412 (1995)). These signal transduction pathways are involved in numerous physiological processes, including the regulation of hormone secretion. It is well established that regulation of intracellular cAMP modulates hormone release in the pancreas (Y. Korman, S. J. Bhathena, N. R. Voyles, H. K. Oie, L. Recant, Diabetes 34, 717 (1985); C. B. Wollheim, Diabetes 29, 74 (1980)). AM has also been reported to have an effect on Na+channel activity (EP Application No. 0 622 458 A2). Since AM has been reported to influence the secretion rate of several hormones, including catecholamine (F. Kato et al., J. Neurochem. 64, 459 (1995); EP Application No. 0 622 458 A2), adrenocorticotropin (W. K. Samson, T. Murphy, D. A. Schell, Endocrinology 136, 2349 (1995)), and aldosterone (T. Yamaguchi, K. Baba, Y. Doi, K. Yano, Life Sci. 56, 379 (1995)), we investigated the potential role of AM in regulating endocrine physiology of the pancreas.
Accordingly, due to the numerous therapeutic and diagnostic applications of AM peptides, there is an enormous medical and health requirement for potent, stable and selective AM peptides for therapeutic uses in the prevention, diagnosis, and treatment of AM related disease and conditions.